Mastitis, an inflammation of the mammary gland that is particularly common in sub-Saharan Africa, including Nigeria, is one of the most economically important conditions affecting the intrinsic quality of ruminant dairy products globally (Halasa et al., 2007; Abebe et al., 2022). Based on the clinical condition of the mammary gland, mastitis is classified into two groups: clinical and subclinical mastitis. Clinical mastitis presents with sudden redness and a painful udder that secretes low-quality milk with higher somatic cell counts (Cobirka et al., 2020). In contrast, subclinical mastitis is characterized by the absence of visible signs in the udder and does not visibly reveal the quality of poor milk (Ruegg, 2017).

Due to their opportunistic tendencies and ubiquitous nature in the environment, coliform bacteria, particularly Escherichia coli (E.coli), Klebsiella spp., and Enterobacter spp., are becoming increasingly implicated in both clinical and subclinical cases as causal agents (Bachaya et al., 2021). The rise of coliforms that produce extended-spectrum beta-lactamase (ESBL) in mastitic milk is a growing concern (Klibi et al., 2019). Particularly when this is connected with toxigenic pathogens such as Staphylococcus aureus, which produces enterotoxins in milk-based products, leading to toxic shock syndrome, food poisoning, and cramps (Galal et al., 2006). These coliforms are resistant to a variety of beta-lactam antibiotics, which limits the available treatment choices and poses serious health hazards to the general public (Iroha et al., 2023).

Because ESBL-producing coliforms can spread through direct contact with infected animals, ingestion of raw or inadequately pasteurized milk, and environmental pollution, their zoonotic potential is especially concerning (Tansawai et al., 2022). Antimicrobial-resistant bacteria can proliferate and spread throughout animal and human populations in Nigeria, mainly through the food chain and through direct contact with farmers in the smallholder and informal dairy systems. This is exacerbated due to inadequate veterinary supervision, poor biosecurity, excessive antibiotic usage, and unsanitary practices (Odetokun et al., 2020).

Mastitis in Nigerian livestock has been extensively studied in small ruminants, especially goat, with data on subclinical mastitis appearing in cattle in the late 2000s (Shittu et al. 2012). The disease has been described as common in Nigeria due to the farming system and poor hygiene around farms, which promote infection of the udder and subsequently low-quality milk and milk-based products. While several microbes have been implicated in the pathogenesis of mastitis, coliforms have been identified as the most notorious group of pathogens with a higher incidence of cow death and agalactia-related culling when compared to other pathogens (Mbuk et al., 2016). Food animals consume 69,455 tonnes of antimicrobials globally (Ardakani et al., 2024), and a significant portion of these antimicrobials goes into the treatment of mastitis in dairy animals (Theodoridou Oxinou et al., 2025). The excessive use of antibiotics in dairy animals is a major cause of antibiotic use in dairy animals. This is further compounded by the fact that up to 20% of subclinical mastitis (SCM) are unrelated to microbial infection (Mdegela et al., 2009), underscoring the need for caution in the administration of antibiotics.

In the context of ruminant mastitis, this study provides a critical framework for addressing the shared burden of antimicrobial resistance (AMR) caused by ESBL-producing pathogens (WHO, 2022). Understanding the prevalence, drivers, and health risks associated with these resistant coliforms in Nigerian livestock systems is essential for developing integrated strategies to safeguard both public health and animal productivity. This study aims to determine the zoonotic and one health implications of ESBL-Producing Coliforms in ruminants with Subclinical Mastitis in Plateau State, Nigeria.

In Plateau State, 287 milk samples were taken from 54 cows, 34 ewes, and 34 does. Of them, the CMT revealed that 79 (27.5%) had subclinical mastitis. Among the ruminant species with SCM, the prevalence rates were 18 (22.8%) in does, 23 (29.1%) in ewes, and 38 (48.1%) in cows. The prevalence of SCM varied significantly among the ruminant groups, according to statistical analysis (χ² = 8.23, p = 0.0163), suggesting that species type affected the chance of SCM occurrence ( Table 1 ).

E. coli was the most commonly isolated coliform among the 79 CMT-positive samples, appearing in 50 (63.3%) of them. Citrobacter freundii (6.3%), K. pneumoniae (21.6%), K. oxytoca (2.5%), and K. aerogenes (6.3%) came next. E. coli was most prevalent in cows (71%), followed by ewes (60.9%) and does (50%), according to the distribution of these germs among the ruminant species. Notwithstanding these variations, the Fisher’s exact tests for the bacteria showed only C. freundii had a marginal statistically significant difference in the prevalence of coliform across the ruminant groups ( Table 2 ). E. coli was isolated from 4 out of the 12 fecal samples of pastoralists examined.

It was determined that 42 (53.2%) of the 79 CMT-positive samples were coliform isolates that produced ESBLs. The most common isolate that produced ESBLs was E. coli (50%), which was followed by K. pneumoniae (21.4%), K. aerogenes (16.7%), C. freundii (9.5%), and K. oxytoca (2.4%). These isolates were found in all three ruminant species, with the ESBL-positive pool consisting of cows (16), ewes (18), and does (8) ( Table 3 ).

Two important ESBL genes, bla _TEM and bla _CTX-M, were found in 46 coliform isolates by PCR analysis. Among all coliform species and ruminant sources, the bla _TEM gene was more common. K. pneumoniae and K. aerogenes from cows and does reveal dual carriage of both genes in several isolates, while E. coli isolates from pastoralists (4 samples) demonstrated 100% carriage of the bla _TEM gene with no detection of bla _CTX-M ( Table 3 ). Using the Maximum Likelihood Method, phylogenetic analysis further verified the bla _TEM ( Figure 1 ) and bla _CTX-M genes’ ( Figure 2 ) genetic relatedness across species.

In this study, we identified ESBL-producing microorganisms implicated in SCM among ruminants, some of which are known to possess zoonotic potential. While direct transmission from animals to humans was not assessed, our findings contribute to the growing body of evidence suggesting that individuals in close contact with livestock may be at increased risk of exposure to antimicrobial-resistant pathogens and resistance genes (Agusi et al.., 2024). This underscores the importance of continued surveillance within a One Health framework (Aworh et al., 2019; Johnson et al., 2007).

This study showed that subclinical mastitis in ruminants was more prevalent than clinical mastitis. This is attributed to the subtle nature of SCM, making it less problematic for farmers as it scarcely affects the physical quality of milk; hence, farmers focus more on clinical mastitis since subclinical mastitis is difficult to detect. As a result, untreated subclinical mastitis festers and has the potential to spread from animal to animal. Cows, Ewes, and Does have a prevalence of 38%, 23%, and 18%, respectively. These figures corroborate earlier findings by Gebrewahid et al. (2012), who reported a prevalence of 18.03% for Does but a higher prevalence of 28.14% for Ewes. This may be connected to Nigeria’s prevalent poor environmental hygiene and farm management practices, which tend to increase the risk of infectious agents spreading among herds in feedlots and during transhumance, a common practice among local pastoralists. In this study, 79 ruminant milk samples with SCM contained 50 coliform isolates identified as E. coli, K. pneumoniae, K. oxytoca, C. freundii, and K. aerogenes. This is in close agreement with Garedew et al. (2012), who identified the primary mastitogens as E. coli, Klebsiella spp., Enterobacter spp., Citrobacter, Serratia, and Proteus. Organic materials, such as bedding and manure, typically contain large amounts of coliform bacteria (environment). Therefore, from an epidemiologic perspective, environmental factors accounted for the majority of pathogen infections in this study. When coliform bacteria come into contact with teat ends, they enter the udder through the teat sphincter. Coliform bacteria either proliferate quickly or go dormant after entering the mammary gland.

E. coli had the highest prevalence rate of 63.3% among all the ruminants sampled, followed by K. pneumoniae with a prevalence of 21.6%, and K. aerogenes with a prevalence of 6.3%. This might be because E. coli is a common commensal organism in the gastrointestinal tract of ruminants and is more readily shed in feces, making it more likely to be isolated during sampling, especially in environments with suboptimal hygiene and management practices. In a cross-sectional study comparable to this one, 54 distinct bacterial species were found in Gondar, Ethiopia. However, the frequently isolated Gram-negative bacterial pathogens were E. coli (29.6%), Pseudomonas aeruginosa (18.5%), and K. pneumoniae (16.7%) (Garedew et al., 2012).

ESBL-producers strains identified in this study carried either bla _CTX-M or bla _TEM. The overall prevalence rate of bla _CTX-M and bla _TEM in ruminant mastitis was 36.96% and 26.08%, respectively. Ali et al. (2016) reported that bla _CTX-M was the predominant ESBL gene detected from bovine mastitis, with a higher prevalence of 77.78% isolates. However, Yu et al. (2019) had a contrary result where bla _TEM was the most frequently detected resistance gene with 83.1% prevalence in bovine mastitis, followed by bla _CTX-M with 66.3% prevalence.

The resistance gene, bla _TEM, has increasingly been identified in many different sources, including humans, animals, and the environment. During the last decade, it has virtually displaced the other ESBLs within Enterobacteriaceae (Canton et al., 2012). Among 12 stool samples collected from pastoralists, only four E. coli were isolated, and all were bla _TEM. This is significant since it reflects AMR in human isolates, and this may reflect likely transmission of resistant strains or resistance genes from humans to animals or vice versa, particularly in environments with close animal-human contact. Despite the small sample size, this raises concerns about the likely reservoir of resistance genes among the population in contact with livestock.

According to phylogenetic analysis, some of the bla _CTX-M and bla _TEM gene sequences from the ruminants in this study shared ancestry with human genes from various geographical locations. Due to the high level of phylodiversity found in ruminants, clones with different genetic backgrounds may be responsible for the transmission of coliforms carrying the bla _CTX-M and bla _TEM genes (Jena et al., 2017). Surprisingly, none of the samples screened in this study revealed the presence of the bla _SHV gene. This trend was found in Yang et al., 2018 where bla _SHV was not detected, while bla _CTX-M and bla _TEM had 97.26% and 71.23% of isolates in cows with mastitis. The absence of bla _SHV genes in this study may be due to the difference in type and volume of consumption of antibiotics and the difference in time in which the isolates were collected (Al-Agamy et al., 2009). Also, it has become evident that once a bla _CTX-M type enters an area, it becomes prevalent, replacing bla _TEM and bla _SHV as the dominating ESBL (Canton et al., 2012).

This study highlights the One Health implications of ESBL-producing coliforms in Nigerian ruminants with SCM. The veterinary, medical, and environmental sectors must work together to address this issue in order to stop the spread of resistance and protect animal and human populations.

This study emphasizes the zoonotic threat posed by (ESBL)-producing coliform bacteria in Nigeria as well as the public health importance of SCM in ruminants. The hidden but significant danger of AMR transmission from animals to people is highlighted by the high prevalence of ESBL-producing E. coli and Klebsiella species, especially those carrying the bla _TEM and bla _CTX-M genes, in otherwise healthy nursing animals.

There is a significant risk of zoonotic spillover of these resistant infections due to the informal selling and consumption of raw milk, as well as the frequent close human-animal contacts found in Nigeria’s pastoral and smallholder dairy systems. According to the findings, a One Health strategy that encourages better farm hygiene, prudent antibiotic usage, integrated surveillance, and livestock handler education should be put into place.

In the end, combating ESBL-producing organisms in animal health systems is crucial for maintaining the efficacy of vital antibiotics for future generations as well as for protecting animal productivity and reducing the growing threat of AMR to human health.